In recent years, an enormous amount of genetic information has been compiled by genomic analyses of various organisms including humans. These pieces of information represent vast gene libraries that have been created by life. In post-genome researches, there is a strong demand for establishing a technique used to select genetic information for a protein (polypeptide) having the desired function with rapidity and high accuracy. One of the methodologies therefor is the display technology. The display technology refers to a technology by which a gene library is screened to select a nucleic acid that encodes a polypeptide having a particular function, such as the specific binding to a target substance, while a polypeptide responsible for a function has a one to one correspondence with a nucleic acid that encodes said polypeptide.
Ribosome display technology has been developed as a method for bringing the display technology into practical use (patent documents 1-8, non-patent document 1). Ribosome display is a technology used to present a polypeptide in the form of a tripartite complex consisting of mRNA-ribosome-polypeptide formed during translation reaction. By forming the tripartite complex, the correspondence of gene product to genetic information is realized. A target substance and the above-described tripartite complex are contacted with each other, a tripartite complex comprising the desired polypeptide is selected by utilizing the specific binding of the polypeptide to the target substance, and the mRNA contained in this tripartite complex is amplified, whereby the nucleic acid that encodes the desired polypeptide can be acquired. Therefore, to suppress the non-specific binding of the tripartite complex to the target substance and the carrier for binding the same is important in acquiring the nucleic acid that encodes the desired polypeptide with high accuracy and high efficiency.
A cell-free protein synthesis system is normally utilized to form the above-described tripartite complex in ribosome display. The cell-free protein synthesis system is a methodology used to synthesize a protein in vitro by utilizing the factors necessary for protein synthesis contained in cell-extracts such as of Escherichia coli, wheat germ, rabbit reticulocytes, or cultured cells (non-patent document 2). The cell-free protein synthesis system allows the desired protein to be synthesized merely by adding gene (DNA or RNA) for said protein to the reaction system and incubating the system, representing the most convenient among the various methods for acquiring the desired protein. Above all, the method using Escherichia coli extract is most commonly utilized for the reasons of large amounts of protein synthesized and the like. However, cell-extracts also contain a large number of contaminating ingredients that are irrelevant to protein synthesis reaction, posing problems such as the degradation of RNA and protein and the overconsumption of energy (non-patent documents 3 and 4).
A reconstituted cell-free protein synthesis system that has recently been developed by a group including the present inventor is a synthesis system consisting exclusively of specified factors involved in protein synthesis reaction, such as translation factors and ribosome (patent document 9, non-patent document 5). Because the reconstituted cell-free protein synthesis system is a synthesis system prepared by reconstituting independently purified factors, degradation of RNA and enzyme reactions that are irrelevant to protein synthesis reaction, such as metabolic reactions, observed with the use of cell-extracts, are hardly detected. Furthermore, because the composition of the synthesis reaction mixture can be easily adjusted, the reconstituted cell-free protein synthesis system is the cell-free protein synthesis system best-suited for ribosome display (patent documents 10 and 11, non-patent document 6).